p53 and Rb mediate two major tumor suppression pathways that are believed to be functionally inactivated inmost, if not all, human cancers. Understanding how these two pathways are regulated has become a major goal of contemporary cancer cell biology. Along withp53, the ARF-INK4a locus is one of the two most frequently altered loci in human cancer. Functionally, p16INK4a inhibits the activity of cyclin D-dependent kinases (CDK4 and CDK6), thereby maintaining the retinoblastoma protein (Rb) in its growth suppressive state. ARF, on the other hand, mediates an oncogene- activated hyperproliferative checkpoint pathway through binding to and antagonizing the nuclear export of MDM2, thereby preventing cytoplasmic degradation of p53. With a focus on the connection between ARF and p53, I have tried to contribute to our understanding of these two major pathways and thereby cancer development. I had previously discovered that ARF stabilizes p53 through binding to and antagonizing the activity of MDM2-a negative regulator of p53, and thus revealed an ARF-MDM2-p53 tumor suppression pathway. Subsequently, I further elucidated the mechanism of ARFs p53 stabilization: ARF forms nuclear bodies in the nucleoplasm with MDM2 and p53, thereby blocking nuclear export of p53 and preventing its cytoplasmic degradation. I also demonstrated that frequently occurring tumor-derived mutations in the human ARF protein impair its function in blocking p53 nuclear export. More recently, I obtained new evidence showing that: (a) Association of MDM2 with ribosomal protein L5 is necessary for MDM2 nuclear export and this regulation is disrupted by frequent cancer-derived mutations in MDM2. (b) Ribosomal protein L5- binding deficient MDM2 failed to promote p53 degradation but retains its ability to suppress p53's transactivation activity. (c) ARF participates in a multipeptide complex, and (d) MDM2 is efficiently degraded by proteolysis. My current and future studies are aimed at several issues concerning the regulation of ARF-MDM2-p53 pathway: (a) Elucidate the mechanism of p53 and MDM2 nucleo-cytoplasmic shuttling controlled by ribosomal protein L5 and/or ARF. (b) Define the function of ARF-MDM2-p53 nuclear bodies by purifying the ARF complex, and (c) as a long-term goal to identify the mechanism and regulation of MDM2 ubiquitination and degradation. Together these experiments should advance understanding of the regulation of the ARF-MDM2-p53 pathway and the functional consequences of its alterations in human cancer.